Photon induced near field electron microscope and biological imaging system

ABSTRACT

A method of obtaining PINEM images includes providing femtosecond optical pulse, generating electron pulses, and directing the electron pulses towards a sample. The method also includes overlapping the femtosecond optical pulses and the electron pulses spatially and temporally at the sample and transferring energy from the femtosecond optical pulses to the electron pulses. The method further includes detecting electron pulses having an energy greater than a zero loss value, providing imaging in space and time.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Nos. 61/243,963, filed Sep. 18, 2009, entitled “PhotonInduced Near-Field Electron Microscopy,” U.S. Provisional PatentApplication No. 61/346,833, filed May 20, 2010, entitled “Photon InducedNear-Field Electron Microscopy,” and U.S. Provisional Patent ApplicationNo. 61/367,262, filed Jul. 23, 2010, entitled “Biological Imaging with4D Ultrafast Microscopy,” which are commonly assigned, the disclosuresof which are hereby incorporated by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

The U.S. Government has certain rights in this invention pursuant toGrant No. FA9550-07-1-0484 awarded by the Air Force (AFOSR), Grant Nos.CHE0549936 & DMR0504854 awarded by the National Science Foundation, andGrant No. GM 081520 awarded by National Institutes of Health.

BACKGROUND OF THE INVENTION

In materials science and biology, optical near-field microscopies enablespatial resolutions beyond the diffraction limit, but they cannotprovide the atomic-scale imaging capabilities of electron microscopy.Transmission electron microscopy (TEM) is a microscopy technique inwhich a beam of electrons is transmitted through a specimen. An image isformed from the interaction of the electrons and the specimen on animaging device, such as a CCD camera. Despite the advances made in TEMtechniques, there is a need in the art for improved methods and novelsystems for ultrafast electron microscopy.

SUMMARY OF THE INVENTION

According to embodiments of the present invention, methods and systemsrelated to microscopy are provided. In a particular embodiment, methodsand systems are provided to provide for photon induced near-fieldelectron microscopy.

According to an embodiment of the present invention, a system forimaging a sample is provided. The system includes a chamber includingone or more optical ports, a stage assembly disposed in the chamber andadapted to receive a sample to be imaged, and a femtosecond laser sourceoperable to emit an optical pulse along an optical path. The system alsoincludes a first frequency conversion stage disposed along the opticalpath and operable to produce a first frequency converted optical pulseand a cathode optically coupled to the first frequency converted opticalpulse and operable to emit an electron pulse having a predeterminedenergy. The system further includes an electron lens assembly operableto direct the electron pulse to impinge on the sample disposed on thestage assembly and a second frequency conversion stage disposed alongthe optical path and operable to produce a second frequency convertedoptical pulse characterized by a photon energy. The system additionallyincludes an optical system operable to receive the second frequencyconverted optical pulse and direct the second frequency convertedoptical pulse to impinge on the sample disposed on the stage assemblyand a spectral detector operable to capture the electron pulse passingthrough or near the sample. The electron pulse passing through or nearthe sample can have an energy higher than the predetermined energy afterabsorbing energy from the second frequency converted optical pulse.Moreover, the system includes a processor coupled to the detector and anoutput device coupled to the processor.

According to another embodiment of the present invention, a method ofimaging a sample is provided. The method includes providing a first setof femtosecond optical pulses and a second set of femtosecond opticalpulses, directing the first set of femtosecond optical pulses to impingeon a cathode, and generating a set of electron pulses in response to thefirst set of femtosecond pulses. The electron pulses are characterizedby a first energy. The method also includes directing the set ofelectron pulses to impinge on a sample at a first time and directing thesecond set of femtosecond optical pulses to impinge on the sample at asecond time. The difference between the first time and the second timeis less than 2 ps. The method further includes transmitting the set ofelectron pulses through the sample. The set of electron pulsestransmitted through the sample is representative of an image of thesample. The method additionally includes detecting the set of electronpulses transmitted through the sample to provide a data signalassociated with the image of the sample. An energy of the set ofelectron pulses transmitted through or near the sample is higher thanthe first energy after absorbing energy from the second frequencyconverted optical pulse. Moreover, the method includes transmitting thedata signal to an output device.

According to yet another embodiment of the present invention, a methodof obtaining PINEM images is provided. The method includes providingfemtosecond optical pulses and generating electron pulses. The methodalso includes directing the electron pulses towards a sample andoverlapping the femtosecond optical pulses and the electron pulsesspatially and temporally at the sample. The method further includestransferring energy from the femtosecond optical pulses to the electronpulses and detecting electron pulses having an energy greater than azero loss value.

Numerous benefits are achieved by way of the present invention overconventional techniques. For example, embodiments of the presentinvention provide methods and systems suitable for measuring sampleswith nanometer scale spatial resolution and femtosecond scale temporalresolution. Both organic and inorganic samples can be measured usingembodiments of the present invention. As described more fully below,embodiments of the present invention utilize energy gain of electronsresulting from temporal and spatial overlap of the electrons withphotons at a nanostructure. These and other embodiments of the inventionalong with many of its advantages and features are described in moredetail in conjunction with the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified diagram of a photon-induced near-field electronmicroscope system according to an embodiment of the present invention;

FIG. 2A is a plot illustrating temporally resolved electron energyspectra of carbon nanotubes irradiated with an intense femtosecond laserpulse at two different delay times according to an embodiment of thepresent invention;

FIG. 2B is a plot showing a magnified view of the electron energyspectrum obtained at t=0 illustrated in FIG. 2A;

FIG. 3A is a bright-field image of an individual carbon nanotubeobtained using photon-induced near-field microscopy according to anembodiment of the present invention;

FIG. 3B is a plot illustrating the spatial field gradient (of lengthscale L) in image counts according to an embodiment of the presentinvention;

FIG. 3C is a plot of the decay of counts with time according to anembodiment of the present invention;

FIG. 3D shows a series of nine energy-filtered images according to anembodiment of the present invention;

FIG. 4A shows plots of the temporal response of the imaged interfacialfields according to an embodiment of the present invention;

FIG. 4B shows of contour plot of electron counts as a function ofelectron energy and time between the electron and photon pulsesaccording to an embodiment of the present invention.

FIG. 4C is an image taken when the E-field polarization of thefemtosecond laser pulse is parallel to the long-axis of the nanotube;

FIG. 4D is an image taken when the E-field polarization of thefemtosecond laser pulse is perpendicular to the long-axis of thenanotube;

FIG. 5A is a simplified physical depiction of the interaction betweenthe electron, photon, and evanescent field when the electron packetarrives at the nanotube before the femtosecond laser pulse (t<0)according to an embodiment of the present invention;

FIG. 5B is a simplified physical depiction of the interaction betweenthe electron, photon, and evanescent field at the precise moment at t=0when the electron packet, femtosecond laser pulse, and evanescent fieldare at maximum overlap at the carbon nanotube according to an embodimentof the present invention;

FIG. 5C is a simplified physical depiction of the interaction betweenthe electron, photon, and evanescent field during and immediately afterthe interaction (t>0) when the electron gains/loses energy equal tointeger multiples of femtosecond laser photons according to anembodiment of the present invention;

FIG. 6 is a plot illustrating temporally resolved electron energyspectra of a protein vesicle irradiated with an intense femtosecondlaser pulse at two different delay times according to an embodiment ofthe present invention;

FIG. 7A is a bright-field TEM image of protein vesicles;

FIG. 7B is a PINEM image of a protein vesicles according to anembodiment of the present invention;

FIGS. 8A-F are ultrafast high-magnification PINEM images of a proteinvesicle according to an embodiment of the present invention;

FIG. 9A is a bright-field TEM image of a whole unstained and unfixed E.coli cell;

FIG. 9B is a PINEM image of the whole unstained and unfixed E. coli cellaccording to an embodiment of the present invention;

FIGS. 10A-F are ultrafast high-magnification PINEM images of the wholeunstained and unfixed E. coli cell according to an embodiment of thepresent invention;

FIGS. 11A-E are ultrafast high-magnification PINEM images of the wholeunstained and unfixed E. coli cell according to an embodiment of thepresent invention;

FIG. 12 is a simplified schematic diagram illustrating a PINEM systemaccording to an embodiment of the present invention; and

FIG. 13 is a simplified flowchart illustrating a method of obtainingPINEM images according to an embodiment of the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Embodiments of the present invention provide methods and systems forphoton-induced near-field electron microscopy (PINEM). In someembodiments of the PINEM technique, the sample (e.g., a biologicalstructure) is exposed to single-electron packets and simultaneouslyirradiated with femtosecond laser pulses that are coincident with theelectron pulses in space and time. By electron energy-filtering thoseelectrons that gain photon energies, the contrast is enhanced only atthe surface of the structures involved.

The high-magnification PINEM imaging described herein provides nanometerscale resolution and femtosecond temporal resolution. As described morefully below, PINEM utilizes both photons and electrons in a microscopeto provide nanometer spatial and femtosecond temporal resolution withenhanced contrast. This near-field method is selective to fields ofstructures whose dimensions are orders of magnitude smaller than thediffraction limit of the light and provides unique polarization andtemporal features.

Some embodiments of the present invention utilize the filtering ofelectron energy only in the gain region (i.e., when the electronacquires photon energy) rather than the conventional use of the lossregion when the electron gives up its energy to the specimen. Thus, thecontrast “lights up” and the femtosecond temporal response is resolved.Because imaging of evanescent fields enhances the contrast, the spatiallocation of the enhancement can be controlled via laser polarization,time scale, and tomographic tilting. PINEM is applicable to both theimaging of inorganic systems as well as biological systems, despite thedifferences in intrinsic properties of these systems. Thus, embodimentsof the present invention provide methods and systems that are applicableto the imaging of a wide range of both inorganic and organic materials.

According to embodiments of the present invention, discrete electronenergy gain and loss processes are utilized in ultrafast electronmicroscopy. Typically, the passage of an electron through a thinspecimen results in the kinetic energy being either conserved (elastic)with only momentum change or reduced (inelastic) through the excitationof the specimen. In electron microscopy, both types of scatteredelectrons can be used to form an image, the appearance of which dependsupon the specific atomic structure and composition of the specimen. Inaddition, the element-specific electron scattering provides a means togenerate a chemical map of the sample region of interest with highresolution. These types of scattering events produce losses of probingelectron energies, corresponding to the energies of the atomic coreelectrons. The so-called “low-loss region” near the (elastic) zero-lossenergy pertains to those electrons that have lost only a small amount ofkinetic energy, typically because of interactions with valence electronsand bulk and surface plasmons.

According to embodiments of the present invention, and in contrast withthe above description involving the energy loss (or no loss) that formsthe basis for conventional electron microscopy imaging, the gain ofenergy by an electron is used to perform imaging. As described morefully throughout the present specification, the gain of energy by theelectron as a result of interaction between a femtosecond laser pulseand an ultrashort electron packet that are overlapped in space and timeat a nanostructure in situ produces a unique region of energy gain. Theinteraction between the photon and electron at the nanostructure resultsin the 200-keV electrons gaining n quanta of photon energy; in otherwords, the electron absorbs, instead of emitting, the photon energy.Both the absorption and emission of light by the electrons produce peaksin the energy spectrum, and these peaks are located in the gain and lossregions at integer multiples of the photon energy. According to theembodiments described herein, the electrons that have absorbed photonsare selected by energy filtering, and an image can be formed that showsprecisely where the gain events have occurred. Only electrons thattravel near the structure absorb photons, and thus with this filteringin the gain region embodiments reach high resolution in contrast andwithout interference from background because of contrast of the elasticand loss regions—i.e., an enhancement of the nanoscale contrast.

Given the nature of interactions between electrons and photons, andconsidering their connections through nanostructures, the inventors haveachieved imaging of evanescent electromagnetic fields with electronpulses when such fields are resolved in both space (nanometer and below)and time (femtosecond time scale).

According to embodiments of the present invention, the precisespatiotemporal overlap of femtosecond single-electron packets withintense optical pulses at a nanostructure (e.g., an individual carbonnanotube, a silver nanowire, or the like) results in the directabsorption of integer multiples of photon quanta (nω) by therelativistic electrons accelerated to 200 keV. By energy-filtering onlythose electrons resulting from this absorption, embodiments imagedirectly in space the near-field electric field distribution, obtain thetemporal behavior of the field on the femtosecond timescale, and map itsspatial polarization dependence. Embodiments of the present inventionleverage the observation of the photon-induced near-field effect inultrafast electron microscopy to provide systems suitable for manyapplications, including those of direct space-time imaging of localizedfields at interfaces and visualization of phenomena related tophotonics, plasmonics and nanostructures.

Imaging in conventional electron microscopes is based on elasticinteractions of electrons with matter; that is, with no energy loss orgain. With variant techniques, these scattering processes at differentangles provide structural and bonding information from images anddiffraction patterns. As such, electron energy loss spectroscopy (EELS)is a powerful analytical tool. When images, diffraction, or electronspectra are time-resolved in electron microscopy, photons are typicallyused to initiate a change for the study of ultrafast structural dynamicsthat are directly the result of photon-matter interactions, which occuron the femtosecond and longer timescale. But, before these structuralchanges, electronic distributions are altered, with their dynamicalchanges being on the femtosecond and shorter timescale and are thedirect result of Photon-electron interactions.

In free space, an electron cannot absorb a quantum of electromagneticenergy because of the lack of energy-momentum conservation. However,absorption followed by stimulated emission can occur when two(counter-propagating) photons are used. In fact, an intense standingoptical wave can result in momentum transfer to free electrons withscattering rates approaching that of the optical frequency. Thesephoton-electron interactions are basic to attosecond pulse generationand to multi-photon harmonics and the laser-assisted surfacephotoelectric effect. The inventors have determined that the field canbe induced and probed on the ultrashort timescale in order to visualizeand control the field for applications in imaging and spectroscopy.

As described more fully throughout the present specification, imagingand spectroscopy methods are provided that rely on the stronginteraction between photons and electrons. In some implementations,structural dynamics commence after the photon-induced near-field (PIN)effect diminishes (in some implementations after 400 fs), reducing theability to perform imaging of the nanostructure electronic propertiesbeyond this time. On this electron-photon interaction timescale, andusing intense pulses with peak irradiances of the order of 100 GW/cm²,the 200 keV electron packets lose and gain energy in discrete quantathat are integer multiples of the tuned photon energy. As described morefully throughout the present specification, absorption/emission of atleast eight quanta of photon energy can be observed despite the factthat the interaction time with the nanostructure is only a few hundredattoseconds, given the electron speed and path length in thenanostructure. In some implementations, up to 40 or more photons areabsorbed by electrons. Thus, the illustration of 8-photon processes areprovided merely by way of example and multi-photon processes involvingmore or less than 8 photons are include within the scope of the presentinvention. Moreover, by energy-filtering the zero-loss peak (ZLP),embodiments of the present invention provide methods and systems toobtain the images formed as a result of elastic scatterings. In anembodiment that selects only the electrons that have gained quanta ofphoton energy, the evanescent electric field can be visualized in realspace images of the nanostructure. Thus, the field polarization andtemporal behavior are different from those of bulk structuraltransformations.

According to embodiments of the present invention, an individualmultiwalled carbon nanotube with a diameter of ˜140 nm and a length of˜7 μm (Aldrich, >90% purity) is utilized for imaging. In anotherembodiment, collections of these multiwalled carbon nanotubes areutilized. In yet another embodiment, silver nanowires with diameters of˜100 nm and lengths ranging from 2 to 20 μm are utilized for imaging.Other nanostructures can be utilized and these examples are not intendedto limit the scope of the present invention.

FIG. 1 is a simplified diagram of a PINEM microscope system according toan embodiment of the present invention. This diagram is merely anexample, which should not unduly limit the scope of the claims herein.One of ordinary skill in the art would recognize many variations,modifications, and alternatives. The ultra-fast electron microscopeillustrated in FIG. 1 can be operated at 200 kV and provides forrecording of both femtosecond-resolved electron energy spectra andenergy-filtered photon-induced images. In other embodiments, otheracceleration voltages are utilized as appropriate to the particularapplication and the 200 kV operation is merely discussed as an example.As illustrated in FIG. 1, a femtosecond laser 110 is directed through aPockels cell 112, which acts as a controllable shutter. Although afemtosecond laser 110 is illustrated in FIG. 1, it will be appreciatedthat the laser 110 can be fiber oscillator/amplifier laser system or thelike. The femtosecond laser 110 is configured in one embodiment tooutput 1,040 nm light of femtosecond pulses. Although a particularoutput wavelength is described herein, embodiments of the presentinvention are not limited to this particular wavelength. In someembodiments, the femtosecond laser 110 provides a wavelength tunabilityfunction that enables the wavelength of the femtosecond pulses to bemodified in order to provide a range of wavelengths as appropriate tothe particular sample. In other embodiments, multiple lasers are used togenerate the electron generation pulses and/or the clocking signalpulses. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

A Glan polarizer 114 is used in some embodiments, to select the laserpower propagating in optical path 115. A beam splitter (not shown) isused to provide several laser beams to various portions of the system.Although the system illustrated in FIG. 1 is described with respect toimaging applications, this is not generally required by the presentinvention. One of skill in the art will appreciate that embodiments ofthe present invention provide systems and methods for imaging,diffraction, crystallography, electron spectroscopy, and related fields.Particularly, the experimental results discussed below yield insightinto the varied applications available using embodiments of the presentinvention.

As described more fully below, the output from a femtosecond laser 110emitting a train of 220 fs pulses centered at 1,040 nm is split into twoarms, one of which is frequency doubled and used to excite thenanostructure, while the other is frequency tripled or quadrupled andused to generate the electron packets at the photocathode source. Thefemtosecond laser 110 is generally capable of generating a train ofoptical pulses with predetermined pulse width. One example of such alaser system is a diode-pumped mode-locked titanium sapphire(Ti:Sapphire) laser oscillator operating at 800 nm and generating 100 fspulses at a repetition rate of 80 MHz and an average power of 1 Watt,resulting in a period between pulses of 12.5 ns. In an embodiment, thespectral bandwidth of the laser pulses is 2.35 nm FWHM. An example ofone such laser is a Mai Tai One Box Femtosecond Ti:Sapphire Laser,available from Spectra-Physics Lasers, of Mountain View, Calif. Inalternative embodiments, other laser sources generating optical pulsesat different wavelengths, with different pulse widths, and at differentrepetition rates are utilized. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

A first portion of the output of the femtosecond laser 110 is coupled toa frequency conversion device 116 that frequency doubles, triples,quadruples, or the like, the train of optical pulses to generate atrain, in an embodiment, of 400 nm, 100 fs optical pulses at an 80 MHzrepetition rate. In the illustrated embodiment, the frequency conversiondevice is a frequency tripling device, thereby generating an opticalpulse at UV wavelengths. In other embodiments, a frequency quadruplingdevice, or other frequency conversion device is utilized. Of course, thedesired output wavelength for the optical pulse will depend on theparticular application. The tripled optical pulse produced by thefrequency conversion device propagates along electron generating path118.

A cw diode laser 120 is combined with the frequency tripled opticalpulse using beam splitter 122. The light produce by the cw diode laser,now collinear with the optical pulse produced by the SHG device, servesas an alignment marker beam and is used to track the position of theoptical pulse train in the electron generating path. The collinear laserbeams enter chamber 130 through entrance window 132. In the embodimentillustrated in FIG. 1, the entrance window is fabricated from materialswith high transparency at the appropriate wavelength, for example, 400nm, and sufficient thickness to provide mechanical rigidity. Forexample, BK-7 glass about 6 mm thick with anti-reflection coatings, e.g.MgF₂ or sapphire are used in various embodiments. One of ordinary skillin the art would recognize many variations, modifications, andalternatives.

An optical system, partly provided outside chamber 130 and partlyprovided inside chamber 130 is used to direct the frequency tripledoptical pulse train along the electron-generating path 134 inside thechamber 130 so that the optical pulses impinge on cathode 140. Asillustrated, the optical system includes mirror 144, which serves as aturning mirror inside chamber 130. In embodiments of the presentinvention, polished metal mirrors are utilized inside the chamber 130since electron irradiation may damage mirror coatings used on someoptical mirrors. In a specific embodiment, mirror 144 is fabricated froman aluminum substrate that is diamond turned to produce a mirrorsurface. In some embodiments, the aluminum mirror is not coated. Inother embodiments, other metal mirrors, such as a mirror fabricated fromplatinum is used as mirror 144.

In an embodiment, the area of interaction on the cathode was selected tobe a flat 300 μm in diameter. Moreover, in the embodiment illustrated,the frequency tripled optical pulse was shaped to provide a beam with abeam waist of a predetermined diameter at the surface of the cathode. Ina specific embodiment, the beam waist was about 50 μm. In alternativeembodiments, the beam waist ranged from about 30 μm to about 200 μm. Ofcourse, the particular dimensions will depend on the particularapplications. The frequency tripled optical pulse train was steeredinside the chamber using a computer controlled mirror in a specificembodiment.

In a specific embodiment, the optical pulse train is directed toward afront-illuminated photocathode where the irradiation of the cathode bythe laser results in the generation of electron pulses via thephotoelectric effect. Irradiation of a cathode with light having anenergy above the work function of the cathode leads to the ejection ofphotoelectrons. That is, a pulse of electromagnetic energy above thework function of the cathode ejects a pulse of electrons according to apreferred embodiment. Generally, the cathode is maintained at atemperature of 1000 K, well below the thermal emission thresholdtemperature of about 1500 K, but this is not required by the presentinvention. In alternative embodiments, the cathode is maintained at roomtemperature. In some embodiments, the cathode is adapted to provide anelectron pulse of predetermined pulse width. The trajectory of theelectrons after emission follows the lens design of the TEM, namely thecondenser, the objective, and the projector lenses. Depending upon theembodiment, there may also be other configurations.

In the embodiment illustrated, the cathode is a Mini-Vogel mount singlecrystal lanthanum hexaboride (LaB₆) cathode shaped as a truncated conewith a flat of 300 μm at the apex and a cone angle of 90°, availablefrom Applied Physics Technologies, Inc., of McMinnville, Oreg. As isoften known, LaB₆ cathodes are regularly used in transmission andscanning electron microscopes. The quantum efficiency of LaB₆ cathodesis about 10⁻³ and these cathodes are capable of producing electronpulses with temporal pulse widths on the order of 10⁻¹³ seconds. In someembodiments, the brightness of electron pulses produced by the cathodeis on the order of 10⁹ A/cm²/rad² and the energy spread of the electronpulses is on the order of 0.1 eV. In other embodiments, the pulse energyof the laser pulse is reduced to about 500 pJ per pulse, resulting inapproximately one electron/pulse

Generally, the image quality acquired using a TEM is proportional to thenumber of electrons passing through the sample. That is, as the numberof electrons passing through the sample is increased, the image qualityincreases. Some pulsed lasers, such as some Q-switched lasers, reducethe pulse count to produce a smaller number of pulses characterized byhigher peak power per pulse. Thus, some laser amplifiers operate at a 1kHz repetition rate, producing pulses with energies ranging from about 1μJ to about 2 mJ per pulse. However, when such high peak power lasersare used to generate electron pulses using the photoelectric effect,among other issues, both spatial and temporal broadening of the electronpulses adversely impact the pulse width of the electron pulse or packetproduced. In some embodiments of the present invention, the laser isoperated to produce low power pulses at higher repetition rates, forexample, 80 MHz. In this mode of operation, benefits available usinglower power per pulse are provided, as described below. Additionally,because of the high repetition rate, sufficient numbers of electrons areavailable to acquire high quality images.

In some embodiments of the present invention, the laser power ismaintained at a level of less than 500 pJ per pulse to prevent damage tothe photocathode. As a benefit, the robustness of the photoemitter isenhanced. Additionally, laser pulses at these power levels preventspace-charge broadening of the electron pulse width during the flighttime from the cathode to the sample, thus preserving the desiredfemtosecond temporal resolution. Additionally, the low electron countper pulse provided by some embodiments of the present invention (e.g., asingle electron) reduces the effects of space charge repulsion in theelectron pulse, thereby enhancing the focusing properties of the system.As one of skill in the art will appreciated, a low electron count perpulse, coupled with a high repetition rate of up to 80 MHz provided bythe femtosecond laser, provides a total dose as high as one electron/Å²as generally utilized in imaging applications.

In alternative embodiments, other suitable cathodes capable of providinga ultrafast pulse of electrons in response to an ultrafast optical pulseof appropriate wavelength are utilized. In embodiments of the presentinvention, the cathode is selected to provide a work function correlatedwith the wavelength of the optical pulses provided by the SHG device.The wavelength of radiation is related to the energy of the photon bythe familiar relation λ(μm)≈1.24÷hv(eV), where λ is the wavelength inmicrons and hv is the energy in eV. For example, a LaB₆ cathode with awork function of 2.7 eV is matched to optical pulses with a wavelengthof 400 nm (v=3.1 eV) in an embodiment of the present invention. Asillustrated, the cathode is enclosed in a vacuum chamber 130, forexample, a housing for a transmission electron microscope (TEM). Ingeneral, the vacuum in the chamber 130 is maintained at a level of lessthan 1×10⁻⁶ torr. In alternative embodiments, the vacuum level variesfrom about 1×10⁻⁶ torr to about 1×10⁻¹° ton. The particular vacuum levelwill be a function of the varied applications.

In embodiments of the present invention, the short duration of thephoton pulse leads to ejection of photoelectrons before an appreciableamount of the deposited energy is transferred to the lattice of thecathode. In general, the characteristic time for thermalization of thedeposited energy in metals is below a few picoseconds, thus no heatingof the cathode takes place using embodiments of the present invention.

Electrons produced by the cathode 140 are accelerated past the anode 142and are collimated and focused by electron lens assembly 146 anddirected along electron imaging path 148 toward the sample 150. Theelectron lens assembly generally contains a number of electromagneticlenses, apertures, and other elements as will be appreciated by one ofskill in the art. Electron lens assemblies suitable for embodiments ofthe present invention are often used in TEMs. The electron pulsepropagating along electron imaging path 148 is controlled in embodimentsof the present invention by a controller (not shown, but described inmore detail with reference to certain Figures below) to provide anelectron beam of predetermined dimensions, the electron beam comprisinga train of ultrafast electron pulses.

The relationship between the electron wavelength (λ_(deBroglie)) and theaccelerating voltage (U) in an electron microscope is given by therelationship λ_(deBroglie)=h/(2 m₀eU)^(1/2), where h, m₀, e are Planck'sconstant, the electron mass, and an elementary charge. As an example,the de Broglie wavelength of an electron pulse at 120 kV corresponds to0.0335 Å, and can be varied depending on the particular application. Thebandwidth or energy spread of an electron packet is a function of thephotoelectric process and bandwidth of the optical pulse used togenerate the electron packet or pulse.

Electrons passing through the sample or specimen 150 are focused andspatially separated as a function of energy by electron lens assembly152 onto a detector 154, which can include a Gatan Imaging Filter.Although FIG. 1 illustrates two electron lens assemblies 146 and 152,the present invention is not limited to this arrangement and can haveother lens assemblies or lens assembly configurations. In alternativeembodiments, additional electromagnets, apertures, other elements, andthe like are utilized to focus the electron beam either prior to orafter interaction with the sample, or both.

Detection of electrons passing through the sample, includingsingle-electron detection, is achieved in one particular embodimentthrough the use of an ultrahigh sensitivity (UHS) phosphor scintillatordetector 154 especially suitable for low-dose applications inconjunction with a digital CCD camera. In a specific embodiment, the CCDcamera was an UltraScan™ 1000 UHS camera, manufactured by Gatan, Inc.,of Pleasanton, Calif. The UltraScan™ 1000 CCD camera is a 4 mega-pixel(2048×2048) camera with a pixel size of 14 μm×14 μm, 16-bitdigitization, and a readout speed of 4 Mpixels/sec. In order to reducethe noise and picture artifacts, in some embodiments, the CCD camerachip is thermoelectrically cooled using a Peltier cooler to atemperature of about −25° C. The images from the CCD camera wereobtained with DigitalMicrograph™ software embedded in the Tecnai™ userinterface, available from FEI of Hillsboro, Oreg. Of course, there canbe other variations to the CCD camera, cooler, and computer software,depending upon the embodiment.

In order to reduce or eliminate space-charge effects, the systemillustrated in FIG. 1 can be operated in the single-electron regime(0.1-1 electrons per packet at the detector). Typical parameters duringoperation are a repetition rate of 500 kHz, a fluence of 14 mJ/cm² forcarbon nanotubes, and a fluence of 1.2 mJ/cm² for silver nanowires. Aportion of the beam from femtosecond laser 110 is frequency doubledusing frequency doubler 160 to produce a frequency doubled beam 164. Thedoubled beam passes through an optical delay line 162, enters chamber130 through entrance window 166, and reflects off mirror 168 to impingeon sample 150. The time delay (on the femtosecond scale) between thefrequency tripled beam (and thus the electron packet) and the frequencydoubled beam is controlled by a controller (not shown). Optical imagingsystem 170 provides for alignment of the beams and viewing of the sampleon the stage.

In order to initiate dynamic changes in the sample, a clocking pulselaser 180 is provided. Clocking pulses from the clocking pulse laser 180are coupled into the chamber using a beam splitter 182 or other suitableoptical element. The clocking pulse can serve to initiate a dynamicchange in the sample and delay of the clocking pulse enables themeasurements using the overlapping electron and photon pulses to be madeof samples during the period of dynamic change.

In one implementation, the repetition rate of the femtosecond laser wasvaried between 1.4 and 1.55 MHz. PINEM cell images described below wereacquired with a specimen excitation fluence of 1.3 mJ/cm² (50 μm FWHM),and electron energy gain/loss spectra and PINEM images of the proteinvesicle were obtained with an excitation fluence of 5.4 mJ/cm². Asdescribed more fully throughout the present specification, imagesobtained using some embodiments of the present invention are generatedby energy filtering and using only the gain region of the spectrum.After locating the maximum spatiotemporal overlap of the femtosecondlaser pulse and electron packets at the specimen, energy-filtered imagesare provided by setting a slit width of 10 eV and sequentially steppingthe spectrum offset by 1-eV increments until only the gain region isselected (i.e., no part of the ZLP or loss region contributes to theenergy-filtered images). The ZLP intensity as a function of the delaytime provides the temporal response; given the response time of thedielectric protein shell and the optical pulse width (˜250 fs), theobserved response with σ=280±13 fs conclusively indicate the femtosecondnature of the electron pulse and the ultrashort response provided byembodiments of the present invention.

FIG. 2A is a plot illustrating temporally resolved electron energyspectra of carbon nanotubes irradiated with an intense femtosecond laserpulse at two different delay times according to an embodiment of thepresent invention. Referring to FIG. 2A, the zero-loss peak (ZLP) of the200 keV electrons (t=−2 ps curve) is taken when the electron packetarrives before the femtosecond pulse. In this spectrum only the plasmonpeaks are present.

The second curve (t=0 fs curve) was recorded when the electron andphoton pulses were configured for a maximum overlap. The energy spectrumat coincidence of the two pulses (t=0 fs curve) displays different peaksassociated with multiple quanta of photon absorption/emission on boththe lower-energy and higher-energy sides of the ZLP. The insetillustrated in FIG. 2A shows the positive energy gain region multipliedby 5 for the t=0 spectrum, indicating that absorption of at least eightquanta of photon energy can be observed at maximum spatiotemporaloverlap. Embodiments are not limited to eight quanta of photon energyand larger numbers (e.g., up to and exceeding 40 quanta of photonenergy) are included within the scope of the present invention. For thet=−2 ps spectrum, however, the discrete peaks are absent and only the πand π+σ plasmon peaks at 6 and 25 eV, respectively, are observed.

FIG. 2B is a plot showing a magnified view of the electron energyspectrum obtained at t=0 fs illustrated in FIG. 2A. The magnified viewof the electron spectrum illustrated in FIG. 2B reveals that thediscrete peaks on both sides of the ZLP occur at integer multiples ofthe photon energy of the exciting femtosecond pulse (i.e., 2.4 eV inFIG. 2B). From these spectra, it is apparent that the discrete peaksoccur as a consequence of the interaction of the 200 keV ultra-fastelectron packet with the 2.4 eV femtosecond photon pulse. On thistimescale, the large influence of the PIN effect is illustrated by thesubstantial decrease in the ZLP intensity at maximum overlap. From therecorded electron-energy spectra, it was determined that electrons ofthe ultrafast packets can absorb more than eight photons during thebrief interaction with the nanostructure as shown by the inset in FIG.2A. It is important to note that the spectra shown in FIG. 2A and FIG.2B (loss/gain) are observed only in the presence of the nanostructure.The energy is given in reference to the loss/gain of photon quanta bythe electrons with respect to the zero-loss energy. The energy gainpeaks illustrated in FIG. 2B (i.e., to the left of ZLP) can be measuredindividually or in one or more groups. As an example, a different imagecould be formed using each of the energy gain peaks, an image could beformed using all of the energy gain peaks, or some combinations ofsubsets of the energy gain peaks could be used. One of ordinary skill inthe art would recognize many variations, modifications, andalternatives. In some embodiments, the energy gain peaks arecharacterized by a spectral narrowing as a function of electron energy.In some images, filtering of the higher energy peaks may result insharper images as a result. Additionally, as a function of time, thedifferent energy gain peaks may vary in intensity, resulting in imagesfor the individual peak or combinations of the peaks that vary as afunction of time.

On the length scale (d) of the nanostructure, relative to that of thephoton (wavelength λ), the interaction between photons and electrons(that is, free-free transitions) is greatly enhanced by the evanescentfield that is created through the excitations of the carbon nanotube,and similarly for the silver nanowires, without which energy-momentumconservation is not achieved. The probability of electron-photoncoupling in the presence of a third body (for example, atom, molecule,or a surface) increases as the electron energy increases for a fixedlaser intensity and wavelength, and such a characteristic is ideal forthe ultrafast electron microscope operated at 200 keV. Free-freetransitions in the electron continuum, without perturbations from thethird body, become descriptive of the process when the electron has amuch higher energy than the photon. Whereas plasmonic fields induced bythe femtosecond pulse can follow the laser electric field, therelaxation time for metallic nanoparticles is of the order of tens tohundreds of femtoseconds, depending on the damping (recombination)processes involved. Multiwalled carbon nanotubes are metallic in nature,as are silver nanowires, and for such metallic nanostructures absorptionis enhanced at lower energies.

FIG. 3A is a bright-field image of an individual carbon nanotubeobtained using photon-induced near-field microscopy according to anembodiment of the present invention. The photon-induced near-fieldelectron microscope image of the individual nanotube illustrated in FIG.3A is a bright-field image shown for reference (time-averaged,unfiltered). The average diameter across the tube is 147±20 nm and thescale bar is 500 nm. As illustrated in FIG. 3A, embodiments of thepresent invention enable imaging of single structures, such as a singleparticle, a single protein, or the like.

FIG. 3B is a plot illustrating the spatial field gradient (of lengthscale L) in image counts according to an embodiment of the presentinvention. The profile illustrated in FIG. 3B was obtained from the t=0frame. The exponential curve on the right side of the plot is displayedto illustrate the typical length scale of an evanescent field. FIG. 3Cis a plot of the decay of counts with time according to an embodiment ofthe present invention.

FIG. 3D shows a series of nine energy-filtered images according to anembodiment of the present invention. The nine energy-filtered imageswere acquired by using only the electrons that have gained energy (up ton=4) relative to the ZLP. For additional clarity, the images can bedisplayed in false color (see bar at lower right). Darker colorsindicates regions of the CCD where no counts were recorded because weselected only the +nω region. The time of arrival of the electronpacket at the nanotube relative to the clocking laser pulse is shown inthe upper left corner of each image. The electric field of the clockinglaser pulse was linearly polarized perpendicular to the long-axis of thenanotube. The counts are shown by a brighter intensity in FIG. 3D andrepresent the fields created by the femtosecond pulse around the surfaceof the nanostructure and their decay with time.

In FIG. 3D, the effect of energy-filtering on imaging is displayed atdifferent times. By using an energy filter (n=1 to 4; 10 eV total width)to select only those electrons that have absorbed energy to form animage, the evanescent field generated by the femtosecond excitationpulse became evident in real-space images of the isolated (individual)nanotube. Further, by varying the arrival time of the electron packet atthe carbon nanotube relative to the femtosecond optical pulse, theultrafast evolution of the evanescent field was followed in real time.As can be seen in FIG. 3D, image counts appear only within the localvicinity of the surface of the carbon nanotube; no energy gain occursfar from the nanostructure or within the tube itself.

The energy-filtered image generated by selecting only energy-gainedelectrons and obtained at t=−600 fs shows almost no counts. As thetemporal overlap increases, however, the counts due to the evanescentoptical field increases and reaches a maximum at t=0 (that is, maximumoverlap) before decreasing again to almost zero at t=+600 fs. Inaddition to revealing the rise time of the evanescent field to be muchless than one picosecond, the sequence of images in FIG. 3D also showsthat the field extends at the interface to ˜50 nm (1/e) into vacuum oneither side of the nanotube. The image length scale is consistent withtheoretical considerations of optically excited plasmons. The timenecessary for the image counts to decay from the maximum to minimumvalues, normalized by the maximum change in counts per unit time asshown in FIG. 3C is 130 fs, reflecting the rate of change, as discussedbelow.

FIG. 4A shows plots of the temporal response and polarization dependenceof the imaged interfacial fields according to an embodiment of thepresent invention. In FIG. 4A, the temporal dependence of the ZLP andrepresentative peaks of ±nω are plotted on a log scale. Inserted intothe plot are the values for τ_(p) of the femtosecond optical pulse (220fs) and τ_(σ) of the fitted transient (420 fs). The t=0 position isdetermined by the temporal response of the ZLP. The inset in FIG. 4Aillustrates a linear contour plot of the data fitted by the Gaussians.

The ultrafast response shown in the discrete energy gain and loss (thatis, bands on both sides of the ZLP (see FIGS. 3A-3D)) can be quantifiedin energy and time space as illustrated in FIGS. 5A-5C. In order toobtain the intensity profile, each energy spectrum was fitted to aseries of Gaussians having the form:

$\begin{matrix}{{S(E)} = {{\frac{1 - {2\alpha}}{\sqrt{2{\pi\sigma}^{2}}}^{{{- E^{2}}/2}\sigma^{2}}} + {\sum\limits_{\pm n}{\frac{an}{\sqrt{2{\pi\sigma}^{2}}}{^{- E^{2}}/2}\sigma^{2}}}}} & (1)\end{matrix}$

where α is a sum over a_(n), the amplitude of the n^(th) photon process,and σ reflects the energy width. A typical fit of equation (1), whichhas also been invoked in photoelectron studies, to the observed spectrumis shown in FIG. 2B. In FIG. 4A, the temporal dependence of differentsidebands and the ZLP is plotted on a log scale. With Gaussian analysisin the time domain, we obtained the time constants involved. For the±3ω peaks, τ_(σ)=420 fs, which is a direct result of the convolution ofthe femtosecond excitation pulse, the electron packet, and response timeof the evanescent optical field; the femtosecond optical pulse durationτ_(p)=220 fs and energy-filtering may be significant in reducingelectron pulse energy dispersion. All peaks were fitted similarly,giving the range of τ_(σ) to be 510±90 fs. Because of the geometryinvolving the two pulses used in the ultrafast electron microscope usedto collect the data illustrated herein, the (axial) group velocitymismatch is irrelevant here, as it results in a dispersion of ˜1 fs overthe 100 nm path length. The zero loss curve is the top curve in FIG. 4A,the ±2ω curve is the middle curve, and the ±3ω curve is the bottomcurve.

FIG. 4B shows a contour plot of electron counts as a function ofelectron energy and time between the electron and photon pulsesaccording to an embodiment of the present invention. Referring to FIG.4B, the ZLP is characterized by a decreased electron count due to energyabsorption and emission in the vicinity of the nanostructure. Theelectron count is periodic as a result of the different number of quantaof photon energy. As the time between the impingement of electron pulseand the photon pulse increases, the signal drops off. As the timeincreases to several picoseconds, the interaction between the evanescentwave of the photon and the electrons decreases as illustrated in thefigure as the temporal overlap between the femtosecond optical pulse andthe electron pulse decreases.

FIG. 4C is an image taken when the E-field polarization of thefemtosecond laser pulse is parallel to the long-axis of the nanotube.The center of the nanotube is characterized by a low intensity. FIG. 4Dis an image taken when the E-field polarization of the femtosecond laserpulse is perpendicular to the long-axis of the nanotube. Bothpolarization frames were taken at t=0, when the interaction betweenelectron, photons and the evanescent field is at a maximum.

Besides the temporal and energy domain observations, we also examinedthe spatial distribution of the near field from images taken fordifferent polarizations of the femtosecond pulse relative to theorientation of an individual nanotube (or wire). As illustrated in FIGS.4C and 4D, ultrafast electron microscope images were obtained at t=0with the E-field of the femtosecond laser pulse polarized eitherparallel (FIG. 4C) or perpendicular (FIG. 4D) to the long axis of thecarbon nanotube. These frames display striking changes in the images:when the laser polarization is positioned appropriately relative to thenanotube orientation, a spatial enhancement of the evanescent field isobserved in the images. This is because for this case the confinement isin the regime of d<λ. On the other hand, for the other polarization,when d>λ, the tip enhancement is seen when the polarization changes by90°. For this case, certain spatial modes may be formed with uniquedistributions. The precise distribution of the field is dependent uponthe nanoscale geometry of the specimen, and the fact that the apex ofthe tip has a decrease in counts at perpendicular polarization isconsistent with nanometer-scale field calculations.

In addition to analysis of carbon nanotubes, embodiments of the presentinvention are applicable to the analysis of other structures includingsilver nanowires. The inventors have demonstrated that for silvernanowires, similar filtered energy gain images are obtained along withthe electron spectra and the polarization dependence. The irradianceutilized to obtain the images was, however, an order of magnitude lower(10 GW/cm²), consistent with the stronger near-field formed in themetallic nanowire and with the difference in material property. Thepolarization dependence was the same as that of the carbon nanotubes,owing to the similarity of the geometrical structure of the nanotubesand nanowires.

From the above described results, and without limiting embodiments ofthe present invention, PINEM imaging can be illustrated by consideringthe spatiotemporal coordinates of the three bodies involved. FIG. 5A isa simplified physical depiction of the interaction between the electron,photon, and evanescent field when the electron packet arrives at thenanotube before the femtosecond laser pulse (t<0) according to anembodiment of the present invention. In the case illustrated in FIG. 5Ano spatiotemporal overlap has yet occurred. At negative times (t<0), wecan visualize the femtosecond laser pulse as not impinging on thenanostructure, and no discrete (nω) electron energy gain or loss wouldbe observed.

FIG. 5B is a simplified physical depiction of the interaction betweenthe electron, photon, and evanescent field at the precise moment at t=0when the electron packet, femtosecond laser pulse, and evanescent fieldare at maximum overlap at the carbon nanotube according to an embodimentof the present invention. When the laser pulse encounters thenanostructure (t=0), it creates the near-field excitations, and thisinteraction causes the surface field to oscillate with the electricfield of the laser. Because the nanotube diameter (˜140 nm) is much lessthan the wavelength of the light (519 nm), the field is confined (d<2)by the dimensions of the tube (wire), and this confinement sets up anoscillating dipole in the structure. The intensity of the evanescentfield extends beyond the structure of the nanotube and falls offexponentially with distance from the surface. Thus, evanescent fieldseffectively mediate the interaction between the 200 keV electron and the2.4 eV photons in the femtosecond excitation pulse, but theabsorption/emission processes only occur when both the electron andphoton are overlapped in space at the nanostructure and in time at t=0.

FIG. 5C is a simplified physical depiction of the interaction betweenthe electron, photon, and evanescent field during and immediately afterthe interaction (t>0) when the electron gains/loses energy equal tointeger multiples of femtosecond laser photons according to anembodiment of the present invention. The inset in FIG. 5C illustratestwo possible final energies in the continuum due to the free-freetransitions between the imaging electron and the photons in thefemtosecond laser pulse, wherein KE represents kinetic energy.

The orders of magnitude enhancement achieved according to embodiments ofthe present invention may be appreciated when comparing withtime-averaged, CW mode of excitation. For a tightly focused CW laser(10⁶ W/cm²), the number of excitations on the timescale of the field isnearly five orders of magnitude less than achieved by embodiments of thepresent invention using ˜100 GW/cm² irradiance. Further, for CW powersof about 10 W, it would be necessary for the nanostructures to dissipatethe energy without significant structural damage. In someimplementations described herein, the average power is typically on theorder of 100 mW. The precise overlap of pulses according to embodimentsof the present invention allows for signal acquisition times of only afew seconds, as every electron contributes to the gain/loss signal onthe timescale of the field's existence. In contrast, for CW electronspectroscopy, the signal will be overwhelmed by a background whosemagnitude is conditioned by the repetition rate and other factors.Finally, it should be noted that the process of ±nω absorption/emissiondescribed herein takes place for each single-electron, timed packet.

Utilizing embodiments of the present invention, the inventors havedemonstrated that photon-induced near-field imaging with electrons ismade possible by the precise overlap of ultrafast electron packets,intense ultrafast laser pulses, and nanostructures. Thesestructure-mediated (electron-photon interaction) phenomena, as well asthe spatiotemporal properties of the evanescent electric fields, can nowbe imaged in real space and on the femtosecond timescale. By knowing thedistribution of the field and the control over its polarization andtemporal behavior, it is possible to explore the nature of interfacialfields and their role in a variety of applications at the nanoscale ofmaterials. Utilizing embodiments of the present invention, PINEMprovides resolution extending into the domain of ultrafast electronmicroscopy: the atomic scale. Moreover, the systems described herein,which utilize inelastic interactions, the real space images (anddiffraction), which are the result of elastic interactions, can easilybe obtained by removing the energy filter, obviating the need for thescanning requirement of optical near-field methods. Additionally,embodiments of the present invention are applicable to imaging withsub-femtosecond electrons.

FIG. 6 is a plot illustrating temporally resolved electron energyspectra of a protein vesicle irradiated with an intense femtosecondlaser pulse at two different delay times according to an embodiment ofthe present invention. FIG. 6 displays the region of the electron energyspectrum taken when the spatiotemporal overlap of the femtosecond laserpulse and electron packet is optimum (i.e., at a maximum) at a singleprotein vesicle and when they separate temporally by +1 ps (i.e., whenthe electron packet arrives at the vesicle 1 ps after the femtosecondlaser pulse). As can be seen in FIG. 6, the t=0 ps spectrum shows lossand gain peaks occurring at integer multiples of the photon energy,whereas the t=+1 ps spectrum does not, similar to what was observed fornon-biological materials. Whereas all regions of the spectrum can beselected and used to generate images, only those electrons that havegained energy (i.e., the gain region) are used in some embodiments. Asillustrated herein, the elastic and loss regions can be used for imagingof the same specimen, e.g., in the bright-field mode. Thus, the spatialcharacteristics of the gain process can be directly visualized, asdiscussed below. In the inset of FIG. 6, the femtosecond temporalresponse of both the electron and photon pulses is shown as the changein the area of the zero-loss peak (ZLP) as a function of time,illustrating the feasibility of visualizing ultrafast dynamics with thistechnique. The fitted response gives σ=280±13 fs. Photon pulses of 250fs in duration were utilized to obtain the data illustrated in FIG. 6.

As described more fully below, the methods and systems described hereinhave been utilized to image biological specimens with femtosecondtemporal resolution. Imaging of the outer shell of liquid-filled proteinvesicles and the cell structure of whole cells of Escherichia coli, bothof which are not absorbing to the photon energy, and are of low-Zcontrast, has been demonstrated. The spatial location of contrastenhancement is controlled via laser polarization, time resolution, andtomographic tilting.

Embodiments of the present invention are applicable to the PINEM imagingof individual biological structures, biomimetic protein vesicles, andthe like. The results from whole unstained E. coli cells are discussedbelow. FIG. 7A is a bright-field transmission electron microscopy (TEM)image of protein vesicles. FIG. 7B is a PINEM image of a proteinvesicles according to an embodiment of the present invention. Thevesicles are composed of a shell of covalently cross-linked BSA proteinmolecules encapsulating a liquid core and are useful as biomimeticcellular structures as well as for a wide range of practical purposes(e.g., drug delivery and contrast agents). The cross-linking is achievedthrough the formation of interprotein sulfur-sulfur bonds via oxidationof cysteine residues in the BSA molecules, and the structure of theindividual particles is not highly denatured during vesicle formation.Therefore, a 500-nm vesicle with a 50-nm-thick shell will be comprisedof ˜500,000 individual BSA protein molecules, each of which occupies ˜60nm³.

The bright-field (BF) image in FIG. 7A was obtained at a magnificationof 27,500×, whereas the PINEM image in FIG. 7B was obtained at amagnification of 67,000×. The PINEM image was recorded at the maximumspatiotemporal overlap of the femtosecond laser pulse and electronpacket although imaging is possible at other overlaps as describedbelow. The raw PINEM image was filtered for noise removal, and theborder around the vesicles in the BF image results from the slightdefocus of the lens.

As discussed above, a PINEM image of a vesicle is formed by filteringthe electrons such that those that have gained energy because of the PINeffect are used. The image is generated at or around the maximumspatiotemporal overlap of the laser pulse and electron packet (i.e.,t=0). Referring to FIG. 7B, the PINEM imaging of the vesicle producesenhanced contrast at the edge, where the protein shell resides, relativeto the remainder of the field of view. That is, the intensity far fromthe structure visualized in a typical bright-field image, which isresponsible for the weakened contrast, is not present in PINEM. Here theeffect shown in the images is the result of fields created by thedielectric (protein) shell, relative to vacuum, by the femtosecond laserpulse. This evanescent electric field at the surface of the vesicle isunique to nanostructures, and its strength decays exponentially withdistance.

Beyond the enhanced contrast provided by PINEM, embodiments of thepresent invention also allow for the femtosecond temporal response ofthe PIN effect to be directly visualized and controlled. By changing thedelay between the excitation laser pulse and the ultrashort electronpacket impinging on the protein vesicle, the time dependence of theinteraction can be followed as illustrated in FIGS. 8A-F.

FIGS. 8A-F are ultrafast high-magnification PINEM images of a proteinvesicle according to an embodiment of the present invention. Theresponse is remarkably ultrafast, with the PINEM contrast beingsignificantly weaker after only 200 fs and essentially being zero at ±1ps. The timing of the response in the PINEM images is consistent withthat quantified from the ZLP of a single protein vesicle. Clearly,imaging the fields of concern here occurs on ultrashort time scales.

Referring to FIGS. 8A-F, ultrafast, polarization, and high-magnificationPINEM imaging of a single protein vesicle are provided. FIGS. 8A-C arethree PINEM images of the same protein vesicle, but obtained atdifferent points in time (0 fs, +200 fs, and +1 ps). Each image wasacquired at a magnification of 53,000×. Each image was filtered fornoise removal, and the contrast limits are all set to the same range.FIGS. 8D-F are PINEM images of a protein vesicle generated with thefemtosecond laser light linearly polarized in a plane indicated by thedouble-headed arrows (8D and 8E), as well as a PINEM image of a portionof a protein vesicle obtained at high magnification (8F). Thepolarization images were obtained at a magnification of 67,000×, whereasthe high-magnification image was obtained at 200,000×. Each pixel in thehigh-magnification image corresponds to 8.8 Å. The raw images werefiltered for noise removal.

The controllability and high-spatial resolution capabilities of thePINEM technique for biological imaging are displayed in FIGS. 8A-F. Ascan be seen, the spatial location of the PIN effect around the structureis accomplished by changing the orientation of the plane of polarizationof the femtosecond laser pulse with respect to the vesicle orientation.The location of the gain regions appears as diametrically opposedcontrast enhancements, the specific locations of which precisely followthe laser polarization. A high-magnification PINEM image (pixel size=8.8Å) of one side of a protein vesicle demonstrates the potential tovisualize single cellular particles being tens of nanometers in diameter(e.g., ribosomes), but with the accompanying femtosecond temporalresolution and enhanced contrast capabilities afforded by the techniquesdescribed herein. It should be noted that the PINEM signal scales withthe number of laser photons impinging upon the specimen. Because thevesicles do not absorb the 532-nm laser light, substantial fluences canbe used to form images without causing photothermal damage.

To demonstrate the use of PINEM to image biological structures withincreased complexity relative to simple protein vesicles, we imagedwhole unstained and unfixed cells of the common bacteria E. coli.Whereas the E. coli cells are much more complex than a simple vesicle,they are ideal model systems for demonstrating biological imaging withPINEM. One reason for this is that they are prokaryotes and thus lackthe intracellular complexity of eukaryotic cells (e.g., a membrane-boundnucleus, mitochondria, Golgi bodies, etc.). Another reason is that theE. coli cell has been extensively imaged with electron microscopy, andseveral high-resolution studies of the ultrastructure have beenpublished.

FIG. 9A is a bright-field TEM image of a whole unstained and unfixed E.coli cell. FIG. 9B is a PINEM image of the whole unstained and unfixedE. coli cell according to an embodiment of the present invention. Bothimages were obtained at a magnification of 19,000×. The PINEM image inFIG. 9B was filtered for noise removal. (Scale bars, 500 nm). Referringto FIG. 9A, the bright-field image displays the mass-thickness contrastof the cell and with some variations within the cell. The nucleoid(i.e., DNA material) is visible in the upper portion of the cell as adark (thick) region, as are many small particles of ˜20 nm diameter,presumably ribosomes, dispersed throughout the cytoplasm. We alsoobserve the cellular envelope (i.e., the material comprising the outerand cytoplasmic membranes). Indeed, the ˜50 nm gap between the outer andcytoplasmic membranes, which contained the peptidoglycan layer, isvisible.

Referring to FIG. 9B, the PINEM image of whole unstained and unfixedcells was generated by maximizing the spatiotemporal overlap of thefemtosecond laser pulse and electron packet at the specimen. Severalinteresting features of the PINEM images should be noted. Unlike theprotein vesicles discussed above, enhanced contrast is observed at boththe outer and inner regions of the cell, which is because portions ofthe cell are thin relative to the thick liquid-filled vesicles, theresult of which is similar to thickness contrast in bright-field TEM andUEM images. The PINEM images illustrate that electrons passing throughthe thinner regions of the cell experience gains and losses nearintracellular topological features, a result that bodes well for imagingultrastructure with this technique.

FIGS. 10A-F are ultrafast high-magnification PINEM images of the wholeunstained and unfixed E. coli cell according to an embodiment of thepresent invention. These figures provide insight into the time scale ofthe PINEM of the cells. By changing the delay time between thefemtosecond laser pulse and electron packet incident at the cell, thetime dependence of the image was followed, with results similar to thatachieved with the (dielectric) protein vesicles and inorganic conductingmaterials. Again, the response is ultrafast, with the contrast weakeningwithin 200 fs of maximum laser pulse and electron packet overlap. Thus,the observed enhancement is optimum in UEM.

FIGS. 10A-C illustrate pseudocolor PINEM images and FIGS. 10D-Fillustrate the corresponding three-dimensional surface plots of the samecell, but obtained at different points in time (0 fs, +200 fs, and +2ps). Each image was acquired at a magnification of 53,000×, and all werefiltered for noise removal. The contrast limits are set to the samerange for each row of images. (Scale bars, 500 nm).

Additionally, embodiments of the present invention providetomographic-type images and demonstrate the effect of photonpolarization. FIGS. 11A-E are ultrafast high-magnification PINEM imagesof the whole unstained and unfixed E. coli cell according to anembodiment of the present invention. The images illustrated in FIGS. 11Aand 11B are PINEM images taken at different specimen tilt angles, 0 and+30°, respectively. The femtosecond laser light was linearly polarizedin a plane indicated by the double-headed arrows. The polarizationimages were obtained at a magnification of 19,000×. The raw images werefiltered for noise removal. As the tilt angle of the specimen ischanged, the spatial distribution and strength of contrast varies. Inconventional electron tomography, images obtained at different specimentilt angles can be combined to construct 3D images of biologicalmacromolecules, with the added capability of energy filtering forgenerating element-specific 3D maps. The PINEM images obtained atdifferent tilt angles demonstrate that the technique could be used togenerate similar tomographic images, but with the added capabilities ofenhanced contrast and ultrafast temporal resolution.

FIGS. 11C-E display a series of PINEM images obtained at differentspecimen tilt angles, which are shown in the upper right corner of eachframe. The images were obtained at a magnification of 53,000× at themaximum spatiotemporal overlap of the femtosecond laser pulse andelectron packet. The raw PINEM images were filtered for noise removal,and the contrast limits are set to the same range. (Scale bar, 500 nm inall images shown). The polarization effect illustrated is consistentwith the concept of nanoscale directional change of the field.Embodiments of the present invention provide for multi-dimensionalimaging (for example 5 dimensions) including the dimensions of time,space, wavelength, electron energy, polarization, tilt angle, and thelike.

As described above, embodiments of the present invention are useful forproviding contrast enhancement in imaging and techniques useful forimaging of both biomimetic protein vesicles and whole unstained,unsliced, and unfixed cells. Embodiments enable the visualization ofsingle particles of nanometer-scale dimensions, but with the addedcapability of femtosecond temporal resolution. The controllability ofthe imaging of biological structures, through the laser pulsepolarization and specimen tilting, adds two other dimensions forselectivity in imaging.

According to some embodiments, the photon wavelength is varied to mapdifferent dimensions, to further improve the spatial resolution bynear-resonance confinement of the particle field, and the energyresolution for mapping all structures at once. One of ordinary skill inthe art would recognize many variations, modifications, andalternatives.

FIG. 12 is a simplified schematic diagram illustrating a PINEM systemaccording to an embodiment of the present invention. The PINEM system1200 includes a femtosecond laser 1210 and a clocking laser 1214.Optical elements 1212 are utilized to provide multiple laser beams toelectron microscope 1220. As an example, one or more frequencyconversion stages are used to provide doubled, tripled, quadrupled beamsor the like. One or more beam splitters and an optical delay stage canbe used to provide multiple beams arriving at the sample stage 1234 atdifferent times. As described above, the femtosecond laser 1210 can beused to provide a beam for electron beam generation and a beam forsample illumination. The clocking laser 1214 can be used to provide abeam to initiate dynamic processes in the sample.

The electron microscope 1220 includes a cathode used to generate theelectron beam, electron optics 1232 to collimate and focus the electronbeam and to direct the electron beam to impinge on the sample stage1234. Photon optics 1240 can be used to direct the intense femtosecondpulses and/or the clocking pulses inside the chamber of the electronmicroscope. A spectral detector 1236, for example, a Gatan ImagingFilter, is provided to detect electrons that have absorbed energy due totheir interactions with the intense femtosecond pulse.

Controller 1238 interacts with data processor 1250, memory 1252, and theinput/output (I/O) module 1254 to enable a user to control the PINEMsystem and to obtain data and images from the PINEM system. The dataprocessor 1250, the memory 1252, and the I/O module 1254 may be providedas part of a computer system. The data processor 1250 represents acentral processing unit of any type of architecture, such as a CISC(Complex Instruction Set Computing), RISC (Reduced Instruction SetComputing), VLIW (Very Long Instruction Word), or a hybrid architecture,although any appropriate processor may be used. The processor 1250executes instructions and includes that portion of a computer thatcontrols the operation of the entire computer. Although not depicted inFIG. 12, the processor 1250 typically includes a control unit thatorganizes data and program storage in memory and transfers data andother information between the various parts of the computer. Theprocessor 1250 receives input data from the I/O device input module 1254a network (not shown), reads and stores code and data in the memory 1252and presents data to the I/O module 1254. Although the PINEM system 1200is shown to contain only a single processor 1250, the disclosedembodiment applies equally to computers that may have multipleprocessors.

The memory 1252 represents one or more mechanisms for storing data. Forexample, the memory 1252 may include read-only memory (ROM), randomaccess memory (RAM), magnetic disk storage media, optical storage media,flash memory devices, and/or other machine-readable media. In otherembodiments, any appropriate type of storage device may be used.Although only one memory 1252 is shown, multiple storage devices andmultiple types of storage devices may be present. Further, the memory1252 may be distributed across multiple computers, for example on aserver.

The memory 1252 includes a controller (not shown in FIG. 12) and dataitems. The controller includes instructions capable of being executed onthe processor 1250 to carry out the methods described more fullythroughout the present specification. In another embodiment, some or allof the functions are carried out via hardware in lieu of aprocessor-based system. In one embodiment, the controller is a webbrowser, but in other embodiments the controller may be a databasesystem, a file system, an electronic mail system, a media manager, animage manager, or may include any other functions capable of accessingdata items. Of course, the memory 1252 may also contain additionalsoftware and data (not shown), which is not necessary to understand theinvention.

The I/O module 1254 is used to receive input and display output to theuser. The I/O module 1254 may be a liquid crystal display (LCD)well-known in the art of computer hardware. But, in other embodimentsthe I/O module 1254 may be replaced with a gas or plasma-basedflat-panel display or a traditional cathode-ray tube (CRT) display. Instill other embodiments, any appropriate display device may be used.Although only one I/O module 1254 is shown, in other embodiments anynumber of output devices of different types, or of the same type, may bepresent. In an embodiment, the I/O module 1254 displays a userinterface. The I/O module 1254 may also include one or more inputdevices such as a keyboard, mouse or other pointing device, trackball,touchpad, touch screen, keypad, microphone, voice recognition device, orany other appropriate mechanism for the user to input data to the systemand manipulate the user interface previously discussed.

FIG. 13 is a simplified flowchart illustrating a method of obtainingPINEM images according to an embodiment of the present invention. Themethod 1300 includes providing femtosecond optical pulses (1310),generating electron pulses (1312), and directing the electron pulsestowards a sample (1314). The method also includes overlapping thefemtosecond optical pulses and the electron pulses spatially andtemporally at the sample (1316). As described more fully throughout thepresent specification, the spatial and temporal overlap provides fornanometer scale spatial resolution and femtosecond scale temporalresolution. In an embodiments, a time delay between each of the pulsesof the femtosecond optical pulses and a corresponding pulse of theelectron pulses is less than 2 ps. Referring to FIG. 4A, for time delaysgreater than 1 ps, the energy transfer process is decreasing inmagnitude.

The method also includes transferring energy from the femtosecondoptical pulses to the electron pulses (1318) and detecting electronpulses having an energy greater than a zero loss value (1320). As anexample, the zero loss value can be associated with an initial energy ofthe electron pulses (e.g., 200 keV). As described throughout the presentspecification, the energy greater than the zero loss value can includean energy equal to an initial energy of the electron pulses plus one ormore quanta of energy associated with the femtosecond optical pulses.

It should be appreciated that the specific steps illustrated in FIG. 13provide a particular method of obtaining PINEM images according to anembodiment of the present invention. Other sequences of steps may alsobe performed according to alternative embodiments. For example,alternative embodiments of the present invention may perform the stepsoutlined above in a different order. Moreover, the individual stepsillustrated in FIG. 13 may include multiple sub-steps that may beperformed in various sequences as appropriate to the individual step.Furthermore, additional steps may be added or removed depending on theparticular applications. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

It is also understood that the examples and embodiments described hereinare for illustrative purposes only and that various modifications orchanges in light thereof will be suggested to persons skilled in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims.

1. A system for imaging a sample, the system comprising: a chamberincluding one or more optical ports; a stage assembly disposed in thechamber and adapted to receive a sample to be imaged; a femtosecondlaser source operable to emit an optical pulse along an optical path; afirst frequency conversion stage disposed along the optical path andoperable to produce a first frequency converted optical pulse; a cathodeoptically coupled to the first frequency converted optical pulse andoperable to emit an electron pulse having a predetermined energy; anelectron lens assembly operable to direct the electron pulse to impingeon the sample disposed on the stage assembly; a second frequencyconversion stage disposed along the optical path and operable to producea second frequency converted optical pulse characterized by a photonenergy; an optical system operable to receive the second frequencyconverted optical pulse and direct the second frequency convertedoptical pulse to impinge on the sample disposed on the stage assembly; aspectral detector operable to capture the electron pulse passing throughthe sample, wherein the electron pulse passing through the sample has anenergy higher than the predetermined energy; a processor coupled to thedetector; and an output device coupled to the processor.
 2. The systemof claim 1 wherein the chamber comprises a vacuum chamber.
 3. The systemof claim 1 wherein the first frequency conversion stage comprises afrequency quadrupler.
 4. The system of claim 1 wherein the secondfrequency conversion stage comprises a frequency doubler.
 5. The systemof claim 1 wherein the spectral detector is operable to detect electronpulses having energies equal to the predetermined energy plus one ormore quanta of the photon energy.
 6. The system of claim 6 wherein theprocessor comprises an energy filter operable to filter electron pulseshaving energies equal to the predetermined energy plus a number ofquanta of the photon energy.
 7. The system of claim 1 further comprisinga delay stage operable to delay or advance the second frequencyconverted optical pulse.
 8. The system of claim 1 further comprising asecond laser source operable to provide a clocking pulse to the sampleprior to the electron pulse impinging on the sample.
 9. A method ofimaging a sample, the method comprising: providing a first set offemtosecond optical pulses and a second set of femtosecond opticalpulses; directing the first set of femtosecond optical pulses to impingeon a cathode; generating a set of electron pulses in response to thefirst set of femtosecond pulses, wherein the electron pulses arecharacterized by a first energy; directing the set of electron pulses toimpinge on a sample at a first time; directing the second set offemtosecond optical pulses to impinge on the sample at a second time,wherein a difference between the first time and the second time is lessthan 2 ps; transmitting the set of electron pulses through the sample,the set of electron pulses transmitted through the sample beingrepresentative of an image of the sample; detecting the set of electronpulses transmitted through the sample to provide a data signalassociated with the image of the sample, wherein an energy of the set ofelectron pulses transmitted through the sample is higher than the firstenergy; and transmitting the data signal to an output device.
 10. Themethod of claim 9 wherein the difference between the first time and thesecond time is less than 1 ps.
 11. The method of claim 10 wherein thedifference between the first time and the second time is less than 600fs.
 12. The method of claim 9 wherein providing the first set offemtosecond optical pulses comprises frequency quadrupling a set offemtosecond laser pulses.
 13. The method of claim 9 wherein providingthe second set of femtosecond optical pulses comprises frequencydoubling a set of femtosecond laser pulses.
 14. The method of claim 9further comprising aligning a polarization of the second set offemtosecond optical pulses with respect to a longitudinal axis of thesample.
 15. The method of claim 9 wherein the energy of the set ofelectron pulses transmitted through the sample is equal to the firstenergy plus one or more quanta of an energy of the second set offemtosecond optical pulses.
 16. The method of claim 15 wherein detectingthe set of electron pulses transmitted through the sample comprisesenergy filtering the set of electron pulses transmitted through thesample to select pulses having energies equal to the first energy plus anumber of quanta of the energy of the second set of femtosecond opticalpulses.
 17. The method of claim 9 further comprising: providing anoptical clocking pulse; and directing the optical clocking pulse toimpinge on the sample at a time prior to the first time.
 18. A method ofobtaining PINEM images, the method comprising: providing femtosecondoptical pulses; generating electron pulses; directing the electronpulses towards a sample; overlapping the femtosecond optical pulses andthe electron pulses spatially and temporally at the sample; transferringenergy from the femtosecond optical pulses to the electron pulses; anddetecting electron pulses having an energy greater than a zero lossvalue.
 19. The method of claim 18 wherein overlapping the femtosecondoptical pulses and the electron pulses temporally at the samplecomprising a time delay between each of the pulses of the femtosecondoptical pulses and a corresponding pulse of the electron pulses of lessthan 2 ps.
 20. The method of claim 18 wherein the zero loss value isassociated with an initial energy of the electron pulses.
 21. The methodof claim 18 wherein the energy greater than the zero loss valuecomprises an energy equal to an initial energy of the electron pulsesplus one or more quanta of energy associated with the femtosecondoptical pulses.
 22. The method of claim 18 further comprising aligning apolarization of the femtosecond optical pulses normal to a longitudinalaxis of the sample.